Use of gamma-tocopherol and its oxidative metabolite LLU-alpha in the treatment of disease

ABSTRACT

The present invention is generally related to the discovery of the therapeutic benefit of administering γ-tocopherol and γ-tocopherol derivatives. More specifically, the use of γ-tocopherol and racemic LLU-α, (S)-LLU-α, or γ-tocopherol derivatives as antioxidants and nitrogen oxide scavengers which treat and prevent high blood pressure, thromboembolic disease, cardiovascular disease, cancer, natriuretic disease, the formation of neuropathological lesions, and a reduced immune system response are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent applicationSer. No. 09/814,330, filed Mar. 21, 2001, which is a continuationapplication of U.S. patent application Ser. No. 09/461,645, filed Dec.14, 1999, now U.S. Pat. No. 6,242,479, issued Jun. 5, 2001, which is acontinuation application of U.S. patent application Ser. No. 09/215,608,filed Dec. 17, 1998, now U.S. Pat. No. 6,048,891, issued Apr. 11, 2000.All of these prior applications which are hereby expressly incorporatedby reference in their entireties.

FIELD OF INVENTION

[0002] The present invention is generally related to the discovery ofthe therapeutic benefit of administering γ-tocopherol and γ-tocopherolderivatives. More specifically, the use of γ-tocopherol and racemicLLU-α, (S)-LLU-α, or other γ-tocopherol derivatives as antioxidants andnitrogen oxide scavengers which treat and prevent high blood pressure,thromboembolic disease, cardiovascular disease, cancer, natriureticdisease, the formation of neuropathological lesions, and a reducedimmune system response are disclosed.

BACKGROUND OF THE INVENTION

[0003] Vitamin E, an essential fat-soluble vitamin, encompasses eightnaturally occurring compounds in two classes. The first class,tocopherols, have four members designated alpha, beta, gamma and delta.The two major forms, α-tocopherol and γ-tocopherol, differ structurallyonly by a methyl group substitution at the 5-position. The second class,tocotrienols, are molecules related to the tocopherols and also consistof four members designated alpha, beta, gamma and delta. The tocotrienolstructure differs from the tocopherols by possessing three double bondsin their side chain rather than being saturated.

[0004] One of the important chemical features of the tocopherols is thatthey are redox agents which act under certain circumstances asantioxidants. In acting as an antioxidant, tocopherols presumablyprevent the formation of toxic oxidation products, such as perioxidationproducts formed from unsaturated fatty acids. Early on, investigatorsattributed most if not all of the biological activity of the tocopherolsto their ability to act as antioxidants. More recently, however, otherbiological activities have been associated with tocopherols includingthe modulation of signal transduction, modulation of phospholipidmetabolism, inhibition of protein kinase C, inhibition of phospholipaseA and inhibition of prostaglandin production. (Meydani and Mosen, TheLancet 345(8943):170-175 (1995)).

[0005] Further, it has recently been discovered that individual membersin the class of tocopherols may exhibit different biological propertiesfrom one another despite their structural similarity. Someinvestigators, for example, believe that γ-tocopherol, unlikeα-tocopherol, acts in vivo as a trap for membrane-soluble electrophilicnitrogen oxides and other electrophilic mutagens. (Christen et al. Proc.Natl. Acad. Sci. 94: 3217-3222 (1997)). In contrast, others report thatα-tocopherol is a more powerful antioxidant and has ten times thebiological activity of γ-tocopherol. (Meydani and Mosen, The Lancet345(8943):170-175 (1995)). Alpha-tocopherol is also thought to beretained in the body longer than γ-tocopherol and has been shown topreferentially reincorporate into nascent very low-density lipoproteins(LDL). (Christen et al. Proc. Natl. Acad. Sci. 94: 3217-3222 (1997)). Atpresent, an understanding of the differences in biological activity ofthe four tocopherols and their effect on the body is in its infancy.

[0006] Alpha tocopherol is largely considered the most important memberof the class of tocopherols because it constitutes about 90% of thetocopherols found in animal tissues and displays the greatest biologicalactivity in the commonly used bioassay systems. In consequence, vitaminE supplements are almost exclusively made of α-tocopherol and littleinvestigation into the efficacy of supplementation with γ-tocopherol hasbeen conducted.

[0007] The therapeutic benefits of vitamin E supplementation remains asubject of considerable debate. Several studies have proposed thatvitamin E supplementation may prevent a plethora of ills but many ofthese studies fail to provide causal connections between vitamersupplementation and therapeutic benefit; they merely indicate that ahigh dietary or plasma concentration and supplemental intake of vitaminE is associated with a reduced risk of disease. In fact, some studieshave failed to demonstrate that tocopherol supplementation provides anyprotection from disease. (Meydani and Mosen, The Lancet 345(8943):170-175 (1995) and (Christen et al. Proc. Natl. Acad. Sci. 94: 3217-3222(1997)). A reliable method to treat and prevent diseases associated withoxidative stress and vitamin E deficiency is highly desirable.

SUMMARY OF THE INVENTION

[0008] The present invention reveals the discovery of the therapeuticbenefit of administering γ-tocopherol and γ-tocopherol derivatives suchas LLU-α. The novel use of γ-tocopherol and γ-tocopherol derivatives asantioxidants and nitrogen oxide scavengers which treat and prevent highblood pressure, thromboembolic disease, cardiovascular disease, cancer,natriuretic disease, the formation of neuropathological lesions, and areduced immune system response are disclosed.

[0009] One embodiment of the present invention is a medicamentcomprising γ-tocopherol and LLU-α with and without additional activeingredients that are effective in producing a natriuretic effect.Another embodiment is a medicament comprising γ-tocopherol,α-tocopherol, and LLU-α with and without additional active ingredientsthat are effective in producing a natriuretic effect. A furtherembodiment is a medicament comprising γ-tocopherol, β-tocopherol, andLLU-α, with and without additional active ingredients that are effectivein producing a natriuretic effect. Still further, an embodimentcomprising α-tocopherol, γ-tocopherol, β-tocopherol, and LLU-α, with andwithout additional active ingredients that are effective in producing anatriuretic effect, is disclosed. In the alternative, the embodimentsdescribed above may include (S)-LLU-α or other γ-tocopherol derivativesinstead of LLU-α.

[0010] According to the methods of treatment and prevention disclosed,the medicaments described above are administered to subjects sufferingfrom high blood pressure, thromboembolic disease, atherosclerosis,cardiovascular disease, cancer, natriuretic disease, the formation ofneuropathological lesions, and a reduced immune system response. Onemethod involves the administration of a therapeutically beneficialamount of γ-tocopherol, with or without supplementation of LLU-α, tosubjects suffering from a high blood pressure so as to treat and preventthis condition. By another method, a therapeutically beneficial amountof γ-tocopherol, with or without supplementation of LLU-α, isadministered to treat and prevent thromboembolic disease. A relatedmethod to treat and prevent the aggregation of platelets and/or bindingof platelets to adhesive proteins is also disclosed.

[0011] Another method contemplated by the present inventor involves theadministration of a therapeutically beneficial amount of γ-tocopherol,with or without supplementation of LLU-α, to treat and preventcardiovascular diseases, such as ischemia, angina, edematous conditions,artherosclerosis, LDL oxidation, adhesion of monocytes to endothelialcells, foam-cell formation, fatty-streak development, plateletadherence, platelet aggregation, smooth muscle cell proliferation, andreperfusion injury. Further, a method to treat and prevent cancers, suchas lung cancer, prostate cancer, breast cancer, and colon cancer byadministering a therapeutically beneficial amount of γ-tocopherol, withor without supplementation of LLU-α are presented.

[0012] Methods of treatment and prevention of natriuretic diseases, suchas hypertension, high blood pressure, ischemia, angina pectoris,congestive heart failure, cirrhosis of the liver, nephrotic syndrome,ineffective renal perfusion, or ineffective glomerular filtration, byadministering a therapeutically beneficial amount of γ-tocopherol, withor without supplementation of LLU-α are also provided. Additionally,methods of treating and preventing neurological diseases includinghyporeflexia, opthalmoplegia, and axonal dystrophy using atherapeutically beneficial amount of γ-tocopherol, with or withoutsupplementation of LLU-α, are described. Finally, methods to improve asubject's immune system response and a related method to reduce theproduction of free-radicals by administering a therapeuticallybeneficial amount of γ-tocopherol, with or without supplementation ofLLU-α, is revealed.

CHEMICAL STRUCTURE OF LLU-α

[0013] FORMULA A shows the structural formula of LLU-α.

[0014] FORMULA B shows the structural formula of (S)-LLU-60 .

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] In accordance with the present invention, a novel method for thetreatment and prevention of high blood pressure, thromboembolic disease,atherosclerosis, cancer, natriuretic disease, the formation ofneuropathological lesions, and a reduced immune system response isprovided. The method involves administering orally or parenterallysubstantially pure γ-tocopherol or a formulation comprising γ-tocopheroland racemic LLU-α, (S)-LLU-α, or other γ-tocopherol derivative.

[0016] By “LLU-α” is meant the compound6-hydroxy-2,7,8-trimethylchroman-2-propanoic acid, molecular weight of264.1362 and molecular formula of C₁₅H₂₀O₄. LLU-α may be in the racemicform or as the S enatiomer (also denoted as (S)-LLU-α). A generaldiscussion of the isolation and characterization of LLU-α is provided byWechter et al. (U.S. patent application Ser. No. 08/290430) thedisclosure of which is hereby incorporated by reference.

[0017] By “γ-tocopherol derivative” is meant γ-tocopherol metabolitesand synthetic chroman derivatives including, but not limited to, LLU-α,LLU-γ, racemic chromans, chroman methyl esters, chroman esters, chromanamides, R₄ chroman esters, oxidized chroman derivatives, racemic2,5,7,8-tetramethyl-2-(β-carboxyethyl)-6-hydroxy chroman,2,5,7,8-tetramethyl-2-(β-carboxyethyl)-chroman,2,7,8-trimethyl-2-(β-carboxyethyl) chroman, racemic4-methyl-6-(5,6-dimethylbenzohinoyl)-4-hexanolid,4-Methyl-6-(3,5,6-trimethylbenzochinoyl)-4-hexanolid,(S)-4-Methyl-6-(5,6-dimethylbenzochinoyl)-4-hexanolid,2,7,8-Trimethyl-2-(β-carboxyethyl)-6-acetyl chroman,2,7,8-Trimethyl-2-(β-carboxyethyl)-6-acetyl chroman methyl ester, andbenzodipyran methyl ester. Many γ-tocopherol derivatives are natriureticcompounds but the meanining of “γ-tocopherol derivative” is not intendedto be limited to only natriuretic compounds. Other γ-tocopherolmetabolites and synthetic chroman derivatives may be known by those ofskill in the art or will be discovered in the future and are encompassedby this definition.

[0018] By “natriuretic disease” is meant diseases associated withabnormal excretion of sodium from the body. The term natriuretic diseaseincludes but is not limited to hypertension, high blood pressure,ischemia, angina pectoris, congestive heart failure, cirrhosis of theliver, nephrotic syndrome, ineffective renal perfusion, and ineffectiveglomerular filtration, or any combination thereof. Other forms ofnatriuretic disease will be apparent to those of skill in the art andare encompassed by the definition as used in this invention.

[0019] As used herein, the term “natriuretic compound” refers to acompound which increases the rate of sodium excretion withoutcontributing to significant potassium loss in a mammal uponadministering the compound to the mammal. The term “natriureticcompound” also refers to both the native compound and in vitro or invivo modifications which retain natriuretic activity. It is understoodthat limited modifications, substitution or deletions of functionalgroups may be made without destroying the biological activity. Moreover,it will be recognized by those skilled in the arts of chemistry andpharmaceutical preparation that many derivatives can be made which arebiologically and chemically equivalent to, or even more active than, theindicated compounds hereinafter. Examples of equivalent compoundsinclude esters, ethers, amides and salts of the foregoing compounds.

[0020] “Substantially purified,” when used to describe the state of thenatriuretic compound, denotes the compounds essentially free ofproteins, steroids, and other material normally associated or occurringwith natriuretic compounds in its native environment.

[0021] As used herein, the term “post salt peak” refers to materialeluted from a G-25 Sephadex column which appears immediately after thesodium, potassium, urea and creatinine containing fractions which hasuv. absorbance at 290 nm.

[0022] A material is “biologically active” if it is capable ofincreasing natriuresis in an in vivo assay as described herein.

[0023] By “thromboembolic disease” is meant diseases characterized byplatelet aggregation, platelet adhesion to adhesive proteins, orplatelet hyperactivity. Although thromboembolic disease is commonlyassociated in insulin-dependent diabetic patients, this understanding isnot intended to limit the invention. Elderly patients and patientssuffering from various forms of cardiovascular disease exhibit plateletaggregation, platelet adhesion to adhesive proteins, and platelethyperactivity which can be defined as forms of thromboembolic diseasefor the purposes of this invention. Other forms of thromboembolicdisease will be apparent to those of skill in the art and areencompassed by the definition used in this invention.

[0024] By “cardiovascular disease” is meant diseases associated with thecardio-pulmonary and circulatory systems including but not limited toischemia, angina, edematous conditions, artherosclerosis, LDL oxidation,adhesion of monocytes to endothelial cells, foam-cell formation,fatty-streak development, platelet adherence, and aggregation, smoothmuscle cell proliferation, reperfusion injury, and other conditionsknown by those of skill in the art to be related to the pathogenesis ofcardiovascular disease.

[0025] By “cancer” is meant diseases that have been associated withmutagenesis, cell transformation, oncogenesis, neoplasia, or metastasis,including but not limited to, various forms of lung cancer, prostatecancer, breast cancer, and colon cancer, or any combination thereof.Other forms of cancer will be apparent to those of skill in the art andare encompassed by the definition used in this invention.

[0026] By “neurological disease” is meant diseases associated with thebrain and nervous system, including but not limited to, hyporeflexia,proprioception, opthalmoplegia, and axonal dystrophy. Other forms ofneurological diseases will be apparent to those of skill in the art andare encompassed by the definition as used in this invention.

[0027] Gamma-tocopherol is a water-insoluble, non swelling amphiphile,as are triglycerides and cholesterol. Thus, many of the processesinvolved in the absorption of lipids are also required for absorption ofγ-tocopherol such as emulsification, solubilization within mixed bilesalt micelles, uptake by the small intestine, packaging withinlipoprotein particles, and secretion into the circulation via thelymphatic system. Gamma-tocopherol is transferred to tissues in much thesame manner as other lipids and spontaneous transfer and exchange oftocopherol between cell membranes has been documented. Sinceγ-tocopherol is rapidly absorbed in the lipids of various tissuesincluding the liver, its antioxidant and radical scavenger activitiesprimarily occur in the lipid phase and only tangentially in the aqueousphase. LLU-α, on the other hand, is considerably more hydrophilic thanγ-tocopherol and acts as an antioxidant, a natriuretic compound, andradical scavenger in primarily the aqueous phase. Thus, the presentinventor contemplates a method to treat and prevent disease whichemploys supplements comprising γ-tocopherol with and withoutfortification with racemic LLU-α, (S)-LLU-α, or other γ-tocopherolderivative so as to selectively provide natriuretic redox agents to thelipid and aqueous phases of a patient's body.

[0028] The preparation of soft gelatin capsules comprising commerciallyavailable γ-tocopherol in doses of 200 to 800 mg is understood by thoseof skill in the art. The γ-tocopherol may be present as the free alcoholor the acetate or succinate ester. A supplement of γ-tocopherolpreferably contains at least 60-65% (weight to weight) γ-tocopherol andup to 10% α-tocopherol and 25% β-tocopherol as isolated from soy oil, orin certain circumstances up to 25% δ-tocopherol. Particularly preferredcompositions include at least 70% γ-tocopherol. These formulations areonly intended to guide one of skill in the art and formulations ofγ-tocopherol that would be effective for use in the disclosed methodsmay include as low as 50% (weight to weight) γ-tocopherol or up to 100%(weight to weight) γ-tocopherol, but desirably contain 55% (weight toweight) γ-tocopherol to 95% (weight to weight) γ-tocopherol.

[0029] In another embodiment of this invention, soft gelatin capsulescomprising commercially available γ-tocopherol are fortified with anatriuretic compound such as LLU-α, (S) LLU-α, or other γ-tocopherolderivative some of which may be present as the free acid or a simpleester. One aspect of the invention, for example, comprises a natriureticcompound having the formula I:

[0030] in which

[0031] R is O, S, SO, SO₂, a secondary or tertiary amine group, aphosphate group, a phosphoester group, or an unsubstituted orsubstituted methylene group,

[0032] R₁ and R₂ independently are H, OH, alkyl, aryl, alkenyl, alkynyl,aromatic, ether, ester, unsubstituted or substituted amine, amide,halogen or unsubstituted or substituted sulfonyl, or jointly complete a5- or 6-member aliphatic or aromatic ring,

[0033] R₃ and R₄ independently are H, OH, alkyl, aryl, alkenyl, alkynyl,aromatic, ether, ester, unsubstituted or substituted amine, amide,halogen or unsubstituted or substituted sulfonyl, or jointly complete a5- or 6-member aliphatic, aromatic or heterocyclic ring,

[0034] R₅ is H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ester orunsubstituted or substituted amine,

[0035] R₆ is COOH, COOR₇, CONH₂, CONHR₇, CONR₇R₈, NH₂, NHR₇, NR₇R₈, or acarboxylate salt,

[0036] R₇ and R₈ independently are unsubstituted or substituted alkyl,aryl, alkaryl, aralkyl, alkenyl or alkynyl,

[0037] n is 0 to 3, and

[0038] m is 0 to 5.

[0039] As used herein, the term “substituted” denotes the presence ofone or more substituent such as alkyl, aryl, alkaryl, aralkyl, ether orhalogen. More particular substituents include C₁₋₆ unbranched orbranched alkyl, such as methyl, ethyl, propyl, n-butyl, sec-butyl andtert-butyl, and C₆₋₁₂ aryl, particularly phenyl.

[0040] In a preferred embodiment, R is O. Also preferably, n=1.Preferably, m=2.

[0041] R₆ preferably is COOH.

[0042] Preferably, R₃ is H or OH. Also preferably, R₄ is H or CH₃.

[0043] In a preferred embodiment, R₁, R₂ and R₅ are CH₃.

[0044] Exemplary preferred compounds of formula I include those in whichR is O, R₁, R₂ and R₅ are CH₃, R₃ is OH, R₄ is H or CH₃, R₆ is COOH, n=1and m=2.

[0045] Other exemplary preferred compounds of formula I includes thosein which R is O, R₁, R₂ and R₅ are CH₃, R₃ is H, R₄ is H or CH₃, R₆ isCOOH, n=1 and m=2.

[0046] In a preferred embodiment, R₇ is a C₁₋₆ alkyl group, inparticular CH₃.

[0047] In another preferred embodiment, R₃ is OH.

[0048] Compounds used in the present invention can also be obtained bymodifying the above recited formula in numerous ways while preservingnatriuretic activity. Examples of such active derivatives includecompounds of formulae II-V, below.

[0049] In all formulae described herein, moieties having likedesignations are considered to correspond to each other as like moietiesin related compounds.

[0050] Another aspect of the invention comprises natriuretic compoundshaving the formula II:

[0051] wherein

[0052] R₁ and R₂ independently are H, OH, alkyl, aryl, alkenyl, alkynyl,aromatic, ether, ester, unsubstituted or substituted amine, amide,halogen or unsubstituted or substituted sulfonyl, or jointly complete a5- or 6-member aliphatic or aromatic ring,

[0053] R₃ and R₄ independently are H, OH, alkyl, aryl, alkenyl, alkynyl,aromatic, ether, ester, unsubstituted or substituted amine, amide,halogen or unsubstituted or substituted sulfonyl, or jointly complete a5- or 6-member aliphatic, aromatic or heterocyclic ring,

[0054] R₅ is H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ester orunsubstituted or substituted amine,

[0055] R₆ is COOH, COOR₇, CONH₂, CONHR₇, CONR₇R₈, NH₂, NHR₇, NR₇R₈, or acarboxylate salt,

[0056] R₇ and R₈ independently are unsubstituted or substituted alkyl,aryl, alkaryl, aralkyl, alkenyl or alkynyl,

[0057] R₉ is hydroxyl or unsubstituted or substituted alkoxyl,

[0058] n is 0 to 3, and

[0059] m is 0 to 5.

[0060] In a preferred embodiment, R₁, R₂ and R₅ are CH₃. Preferably, R₃is OH.

[0061] R₄ preferably is H.

[0062] Additionally, it is preferred that n=1. Preferably m=2.

[0063] In a preferred embodiment, R₆ is COOCH₂CH₃ and R₉ is OH. Inanother preferred embodiment, R₆ is COOH and R₉ is CH₃CH₂O.

[0064] Specific examples includes the following:

[0065] A further aspect of the invention comprises natriuretic compoundshaving the formula III:

[0066] wherein

[0067] R₁ and R₂ independently are H, OH, alkyl, aryl, alkenyl, alkynyl,aromatic, ether, ester, unsubstituted or substituted amine, amide,halogen or unsubstituted or substituted sulfonyl, or jointly complete a5- or 6-member aliphatic or aromatic ring,

[0068] R₃ and R₄ independently are H, OH, alkyl, aryl, alkenyl, alkynyl,aromatic, ether, ester, unsubstituted or substituted amine, amide,halogen or unsubstituted or substituted sulfonyl, or jointly complete a5- or 6-member aliphatic, aromatic or heterocyclic ring,

[0069] R₅ is H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ester orunsubstituted or substituted amine,

[0070] n is 0 to 3, and

[0071] q is 0 to 4.

[0072] In preferred embodiments, n=1. Also preferred are compounds inwhich m=2.

[0073] Exemplary natriuretic compounds of formula m include thefollowing:

[0074] The instant invention comprises other natriuretic compoundshaving the formula IV:

[0075] wherein

[0076] R₁ and R₂ independently are H, OH, alkyl, aryl, alkenyl, alkynyl,aromatic, ether, ester, unsubstituted or substituted amine, amide,halogen or unsubstituted or substituted sulfonyl, or jointly complete a5- or 6-member aliphatic or aromatic ring,

[0077] R₄ is H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ether,ester, unsubstituted or substituted amine, amide, halogen orunsubstituted or substituted sulfonyl,

[0078] R₅ is H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ester orunsubstituted or substituted amine,

[0079] R₆ is COOH, COOR₇, CONH₂, CONHR₇, CONR₇R₈, NH₂, NHR₇, NR₇R₈, or acarboxylate salt,

[0080] R₇ and R₈ independently are unsubstituted or substituted alkyl,aryl, alkaryl, aralkyl, alkenyl or alkynyl,

[0081] R₉ is hydroxyl or unsubstituted or substituted alkoxyl,

[0082] n is 0 to 3, and

[0083] m is 0 to 5.

[0084] Preferably n=1. Also, preferably m=2.

[0085] Specific compounds of the invention according to formula IVinclude:

[0086] Natriuretic compounds of formula V are also combined withγ-tocopherol to make the medicaments of the instant invention:

[0087] wherein

[0088] R₁ and R₂ independently are H, OH, alkyl, aryl, alkenyl, alkynyl,aromatic, ether, ester, unsubstituted or substituted amine, amide,halogen or unsubstituted or substituted sulfonyl, or jointly complete a5- or 6-member aliphatic or aromatic ring,

[0089] R₄ is H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ether,ester, unsubstituted or substituted amine, amide, halogen orunsubstituted or substituted sulfonyl,

[0090] R₅ is H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ester orunsubstituted or substituted amine,

[0091] n is 0 to 3, and

[0092] q is 0 to 4.

[0093] Preferred embodiments are those in which n=1. Also, it ispreferred that m=2.

[0094] Included in the inventive compounds of formula V are:

[0095] In accordance with another aspect of present invention,medicaments having the formula Ia and γ-tocopherol are contemplated.

[0096] in which

[0097] R is O, S, SO, SO₂, a secondary or tertiary amine group, aphosphate group, a phosphoester group, or an unsubstituted orsubstituted methylene group,

[0098] R₁ and R₂ independently are H, OH, alkyl, aryl, alkenyl, alkynyl,aromatic, ether, ester, unsubstituted or substituted amine, amide,halogen or unsubstituted or substituted sulfonyl, or jointly complete a5- or 6-member aliphatic or aromatic ring,

[0099] R₃ and R₄ independently are H, OH, alkyl, aryl, alkenyl, alkynyl,aromatic, ether, ester, unsubstituted or substituted amine, amide,halogen or unsubstituted or substituted sulfonyl, or jointly complete a5- or 6-member aliphatic, aromatic or heterocyclic ring,

[0100] R₅ is H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ester orunsubstituted or substituted amine,

[0101] R₆ is COOH, COOR₇, CONH₂, CONHR₇, CONR₇R₈, NH₂, NHR₇, NR₇R₈, or acarboxylate salt,

[0102] R₇ and R₈ independently are unsubstituted or substituted alkyl,aryl, alkaryl, aralkyl, alkenyl or alkynyl,

[0103] n is 0 to 3, and

[0104] m is 0 to 5.

[0105] In preferred embodiments,

[0106] (i) when R is O, R, R₂ and R₅ are CH₃, R₃ and R₆ are OH, and R₄is H, m=2 to 5;

[0107] (ii) when R is O R₁ is H or CH₃, R₂ is H, CH₃, C(CH₃)₃ orCH(CH₃)₂, R₃ is OH or CH₃COO, R₄ is CH₃ or CH(CH₃)₂, R₅ is H, CH₃ orCH₂CH₃, and R₆ is H, OH, OCH₃, OCH₂CH₃ or NH_(2,) m=1 to 5;

[0108] (iii) when R is O, R₁ and R₅ are CH₃, R₂ and R₄ are H, R₃ is OHor CH₃COO, and R₆ is OH or CH₃O m is not 2;

[0109] (iv) when R is O, R₁, R₂ and R₅ are CH₃, R₃ is OH or CH₃COO, R₄is alkyl having at least two carbon atoms, and R₆ is H, OH or ester,m=1; and

[0110] (v) when R, R₂ and R₅ are methyl, R₃ and R₆ are OH and R₄ isalkyl, m=2.

[0111] Certain medicaments of the present invention comprise natriureticcompounds that have been isolated in substantially pure form. Thenatriuretic compounds are obtained from a variety of sources, includingurine, hypothalamus, adrenal, liver, kidney, plasma, blood and culturedcells. Human uremic urine is the preferred source, although normal humanurine or hypertensive human urine may also be used.

[0112] One of the isolated natriuretic compounds used to make amedicament of the present invention is LLU-α. (See FIGS. 1 and 2). LLU-αhas the following properties: a major ultraviolet absorbance peak atabout 210 nm; a broad secondary peak at about 295 nm; instability indilute base; capability of esterification by reaction with CH₂N₂. Thecompound is capable of increasing sodium excretion in the urine inmammals without a corresponding increase in potassium excretion, anddoes not cause a significant change in mean arterial pressure. Thecompound additionally acts as a cardio-selective free radical scavenger.

[0113] Medicaments of the instant invention also comprise anotherisolated natriuretic compound, named LLU-γ, which has the followingproperties: a major ultraviolet absorbance peak at about 220 nm; asecondary peak at about 268 nm; high instability in the presence of O₂or in dilute base. It is capable of increasing sodium excretion inmammalian urine without a corresponding increase in potassium excretion,although potassium excretion (kaliuresis) may be observed occasionallyafter infusion of the compound into conscious rats. In addition, it doesnot cause a significant change in mean arterial pressure and it shows noinhibition of the sodium pump.

[0114] Natriuretic compounds which comprise the present invention can bepurified by a number of methods, particularly those exemplified herein.In a preferred method within the invention, collected urine is processedby ultrafiltration (≦3 kDa), gel filtration chromatography (G-25) andextraction with isopropanol and diethyl ether. The organic solublematerial is then subjected to sequential high-performance liquidchromatography, while assaying for the natriuretic, activity in vivo.Alternatively, collected urine is extracted with ether, separated byhigh performance liquid chromatography, and fractions are assayed fornatriuretic activity.

[0115] In a further alternative embodiment, the natriuretic compounds inthe medicaments of the present invention can be synthesized usingmethods known to those skilled in the art. One such method is the methoddescribed by J. Weichet et al., Czech. Chem. Commun. 24, 1689-1694(1959), the disclosure of which is hereby incorporated by reference.This method can readily be adapted by one of ordinary skill in the artto provide a method of synthesizing the compounds of the presentinvention. Other methods to synthesize the natriuretic compounds of thepresent invention are disclosed in Wechter et al., Proc. Natl. Acad.Sci. USA 93:6002-6007 (1996) and Kantoci et al., J. Pharmacology andExperimental Therapeutics 282:648-656 (1997) which are herebyincorporated by reference.

[0116] A preferred synthetic method includes the step of reacting acompound of the formula VI:

[0117] in which

[0118] R₁ and R₂ independently are H, OH, alkyl, aryl, alkenyl, alkynyl,aromatic, ether, ester, unsubstituted or substituted amine, amide,halogen or unsubstituted or substituted sulfonyl, or jointly complete a5- or 6-member aliphatic or aromatic ring, and

[0119] R₃ and R₄ independently are H, OH, alkyl, aryl, alkenyl, alkynyl,aromatic, ether, ester, unsubstituted or substituted amine, amide,halogen or unsubstituted or substituted sulfonyl, or jointly complete a5- or 6-member aliphatic, aromatic or heterocyclic ring,

[0120] with a vinyl lactone of the formula VII:

[0121] in which

[0122] R₅ is H, alkyl, aryl, alkenyl, alkynyl, aromatic or ester,

[0123] n is 0 to 3, and

[0124] q is 0 to 4.

[0125] In a preferred embodiment of the foregoing synthesis, R₃ is OH.Preferably, R₄ is not simultaneously OH. A preferred compound of formulaVI is a hydroquinone, for example 2,3-dimethyl-1,4-hydroquinone.

[0126] A preferred vinyl lactone of formula VII isγ-methyl-γ-vinylbutyrolactone (R₅=CH₃, n=1, q=1).

[0127] In carrying out the foregoing reaction, preferably a catalyst isused, such as a metallic or non-metallic salt. Specific types ofcatalyst include non-metallic salts which form complexes with a solvent,particularly a catalyst such as boron trifluoride diethyl etherate.

[0128] In carrying out the foregoing reaction, preferably an aprotic orprotic solvent is employed, in particular an aprotic solvent such asdioxane. The catalyst and/or the vinyl lactone is preferably diluted inthe selected solvent.

[0129] Preferably the synthesis is carried out at an elevatedtemperature, such as 100-110° C.

[0130] In a preferred embodiment, the foregoing reaction mixture isdiluted with an aprotic or protic solvent, particularly an aproticsolvent such as diethyl ether.

[0131] The desired product preferably is obtained from concentratedsupernatant which is purified, for example, using an RP-HPLC column orsilica gel. Preferred eluents for RP-HPLC include mixtures of water,acetonitrile and acetic acid. Preferred solvents for silica gel includeethyl acetate and hexane. Other purification methods, such ascrystallization, can be used. Also, other eluents, such as hexane anddimethyl ketone, can be employed.

[0132] The foregoing synthesis produces a racemic mixture, of whichtypically one enantiomer is active while the second enantiomer is lessactive or inactive. The racemate can be employed in compositionsaccording to the invention, with adjustment of the quantity to accountfor the presence of the inactive enantiomer. Alternatively, the racematecan be resolved using conventional methods, and the active enantiomeridentified and utilized. All enantiomeric forms of the compoundsdescribed herein are specifically contemplated as being within the scopeof the instant invention.

[0133] As a byproduct of the foregoing synthesis, derivative compoundsof formula VIII are produced:

[0134] These compounds can also be employed as natriuretic compoundswhich comprise the medicamnet according to the instant invention.Exemplary compounds of formula VIII include the following benzodipyranderivatives:

[0135] All stereoisomeric forms of the foregoing compounds, includingmeso compounds and diastereomeric pairs, are specifically contemplatedas being within the scope of the instant invention.

[0136] Di-oxidized and/or di-hydrated derivatives of the compounds offormula VIII can be obtained in a manner analogous to those used toobtain compounds of formulae II-V from the compounds of formula I.

[0137] As mentioned previously, natriuretic compounds which comprise themedicaments of the instant invention can be modified by formation ofesters, amides, etc. Esterification can be carried out, for example, byreaction with a solution of a diazoalkane, or with an anhydride or anacyl chloride. Amides can be formed by reaction with ammonia or anamine.

[0138] Natriuretic compounds of formulae II-V can be derived from thecorresponding natriuretic compounds produced by the foregoing method,for example, by oxidation. In a preferred embodiment of this process,when R₄=H, R₅ is not CH₃.

[0139] A preferred oxidizer for the foregoing method is a solution offerric chloride. Other oxidants, such as KMnO₄, SeO₂, CrO₃, H₂O₂,m-chloroperbenzoic acid, Caro acid, OSO₄, HIO₄, potassium ferricyanide,silver chromate or sodium perborate, can also be used.

[0140] Scheme 1 illustrates the relationship between exemplary compoundsof formulae I-V. Note that Scheme 1 depicts the relationships betweenthe S-enantiomers. The same relationships exist between thecorresponding R-enantiomers. A wide variety of natriuretic compoundswithin the scope of the instant invention can be obtained in the mannerillustrated.

[0141] Formulations of medicaments comprising γ-tocopherol and LLU-α,(S)-LLU-α, or other γ-tocopherol derivatives, detailed above, are asfollows. Racemic LLU-α is synthesized or isolated and may be present asthe free acid or a simple ester. Racemic LLU-α is added to the differingconcentrations of γ-tocopherol with or without a suitable filler. Asupplement comprising γ-tocopherol and racemic LLU-α preferably contains5% to 95% (weight to weight) γ-tocopherol mixed with 5% to 95% racemicLLU-α, and may also include other tocopherols. More preferably, thecompositions of this embodiment of the invention include between 25% and60% racemic LLU-α, or still more preferably no more than 50% (weight toweight) racemic LLU-α. A particularly preferred composition includes 26%(weight to weight) racemic LLU-α with the remaining amount of thesupplement being composed of tocopherols and a suitable filler, with atleast 65% of the tocopherols being γ-tocopherol.

[0142] Soft gelatin capsules comprising commercially availableγ-tocopherol are fortified with (S)-LLU-α using the same compositions,above. (S)-LLU-α is synthesized or isolated, as detailed above or in thefollowing examples, and may be present as the free acid or a simpleester. (S)-LLU-α is added to the formulations of the γ-tocopherolsupplements mentioned above with or without a suitable filler. Asupplement comprising γ-tocopherol and (S)-LLU-α preferably contains 5%to 95% (weight to weight) γ-tocopherol mixed with 5% to 95% (S)-LLU-α,and may also include other tocopherols. More preferably, thecompositions of this embodiment of the invention include between 25% and60% (S)-LLU-α, or still more preferably no more than 50% (weight toweight) (S)-LLU-α. A particularly preferred composition includes 26%(weight to weight) (S)-LLU-α with the remaining amount of the supplementbeing composed of tocopherols and a suitable filler, with at least 65%of the tocopherols being γ-tocopherol.

[0143] Alternatively, soft gelatin capsules comprising commerciallyavailable γ-tocopherol are fortified with a γ-tocopherol derivative. Theγ-tocopherol derivative is synthesized or isolated, as detailed above orin the following examples, and may be present as the free acid or asimple ester. An γ-tocopherol derivative is added to the formulations ofthe γ-tocopherol supplements mentioned above with or without a suitablefiller. A supplement comprising γ-tocopherol and a γ-tocopherolderivative preferably contains 5% to 95% (weight to weight) γ-tocopherolmixed with 5% to 95% γ-tocopherol derivative, and may also include othertocopherols. More preferably, the compositions of this embodiment of theinvention include between 25% and 60% γ-tocopherol derivative, or stillmore preferably no more than 50% (weight to weight) γ-tocopherolderivative. A particularly preferred composition includes 26% (weight toweight) γ-tocopherol derivative with the remaining amount of thesupplement being composed of tocopherols and a suitable filler, with atleast 65% of the tocopherols being γ-tocopherol. Other tocopherols canbe included in the formulations, including α-tocopherol, β-tocopheroland δ-tocopherol. In certain circumstances, δ-tocopherol can substitutefor γ-tocopherol in the formulations and methods described herein.

[0144] The preferred method of administering principally γ-tocopherol orthe formulation comprising γ-tocopherol and racemic LLU-α, (S)-LLU-α, orγ-tocopherol derivative is orally via soft gelatin capsules, however,several methods of administering these therapeutics would be within theskill of one in the art. Gamma-tocopherol or the formulations mentionedabove can be administered neat, as mixtures with other physiologicallyacceptable active or inactive materials such as moistening agents,flavoring agents, binding agents, and extenders, as well as othercompounds having pharmacological activities, such as other diureticswhich increase the distal delivery of sodium, other anti-cancertherapeutics, other high blood pressure medicaments, otheranti-hypertensive agents, or other mixtures of tocopherols. It may alsobe administered with physiologically suitable carriers such as, forexample, olive oil, sesame oil, or other lipid. The compounds can beadministered orally or parenterally, for example, by injection.Injection can be subcutaneous, intravenous, or by intramuscularinjection.

[0145] The total daily dose of 200-800 mg can consist of a singleindividual dose or multiple doses given at intervals. Dosages withinthese ranges can also be administered by constant infusion over anextended period of time, usually exceeding 24 hours, until the desiredtherapeutic benefits have been obtained. Amounts of the compoundsdescribed herein which are therapeutically effective against specificdiseases can also be determined through routine investigation.

[0146] The following examples are intended to illustrate, but not limitthe invention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may be alternativelyemployed. In the examples, the following abbreviations are used: EIelectron impact FR furosemide response FT-IR Fourier-transform infraredspectroscopy HPLC high performance liquid chromatography MAP meanarterial pressure MDBK Madin-Darby bovine kidney MS mass spectrometryNMR nuclear magnetic resonance PBS phosphate buffered saline R_(n)natriuretic ratio RP-HPLC reverse-phase high performance liquidchromatography SR sample response UNaV urine concentration of sodium Xurine volume per time

Isolation of Natriuretic Compound EXAMPLE 1

[0147] Human uremic urine was initially processed by ultrafiltration (3kDa) and lyophilization, followed by isolation of the post-salt fractionfrom Sephadex G-25 gel filtration chromatography, following theprocedure of Benaksas et al., Life Sci. 52, 1045-1054 (1993), the entiredisclosure of which is herein incorporated by reference. See Table I(first purification step).

[0148] The crude material was further purified by one of two procedures.One procedure involved four sequential HPLC steps, and the secondprocedure included organic solvent extraction followed by up to fivesequential HPLC steps. Table I summarizes the two methods. TABLE 1Summary of steps used in the chromatographic and extraction isolationprocedures Purification Chromatographic Extraction Step Method MethodFirst 3K ultrafiltration, 3K ultrafiltration, lyophilization and G-25lyophilization and G-25 Second 0.2 M pyridinium acetate Sequentialextraction with pH 5.5/Methanol C₁₈ isopropanol/diethyl ether RP-HPLCyielding soluble compounds Third 1st 0.2 M acetic acid/ 1st 0.2 M aceticacid/ methanol C₁₈ RP-HPLC methanol C₁₈ RP-HPLC Fourth 2nd (modified)0.2 M acetic 2nd (modified) 0.2 M acetic acid/methanol C₁₈ acid/methanolC₁₈ RP-HPLC RP-HPLC^(b) Fifth Isopropanol/hexane^(a) Isocratic 0.2 Macetic acid/methanol^(c) Sixth Isopropanol/hexane silica gel HPLCSeventh 50 mM acetic acid/acetonitrile C₁₈ RP-HPLC^(d)

[0149] 1. Chromatographic Isolation Method

[0150] A four-step sequential HPLC procedure was employed which was amodification of the procedure reported by Benaksas et al., noted above.The first C₁₈ RP-HPLC (Table 1, step 2) was performed on a BeckmanUltrasphere ODS column (10 μm; 21.2×150 mm) eluting at 6 mL/minute witha gradient of 0.2 M pyridinium acetate, pH 5.5 (A) and methanol (B) (80%A:20% B for 22 minutes, a linear gradient to 40% A:60% B over 48minutes, a linear gradient to 100% B over 10 minutes). The column waswashed with 70% toluene:30% methanol, then re-equilibrated at initialconditions for at least 20 minutes. This column wash method wasimplemented in every chromatography employing a methanol eluant. Theeluant was monitored with a Beckman 166 UV detector at 290 nm. Eighty(80) one-minute fractions were collected and dried under reducedpressure in a centrifugal vacuum concentrator.

[0151] Based on bioassay evaluation (see Example 2, below) andchromatographic comparison of previous HPLC runs, fractions 50-80, werecombined for the second RP-HPLC step (Table 1, third step). A BeckmanUltrasphere ODS (C₁₈) column (5 μm; 10×250 mm) was eluted at 2 mL/minutewith a gradient of 0.2 M acetic acid (A), methanol (B) and 70% toluene:30% methanol (C), (60% A:40% B for 5 minutes, a linear gradient to 50%A:50% B over 5 minutes, a linear gradient to 30% A:70% B over 55minutes, a linear gradient to 100% B over 2 minutes, 100% B for 3minutes, 100% C for 8 minutes, 100% B for 7 minutes). The eluant wasmonitored for fluorescence (exc. 310-410 nm; emm. 475-610 nm: Beckman157 detector) and absorbance at 290 nm with a Beckman 168 diode arraydetector. Ultraviolet spectra were collected by diode array at 2 secondintervals over the range of 202-390 nm. Eighty (80) one-minute fractionswere collected.

[0152] As discussed in detail in Example 2, two natriuretically activeisolates (LLU-α and LLU-γ) in particular were identified. The regionencompassing the two natriuretically active isolates was pooled andrechromatographed using a modified acetic acid/methanol gradient for thethird RP-HPLC (Table 1, fourth step). The solvents and column were thesame as the second RP-HPLC above; however, the gradient was changed (60%A:40% B for 5 minutes, a linear gradient to 40% A:60% B over 5 minutes,a linear gradient to 30% A:70% B over 28 minutes, a linear gradient to100% B over 2 minutes, 100% B for 3 minutes, 100% C for 8 minutes) andonly fifty (50) one-minute fractions were collected.

[0153] During the first aqueous acetic acid-methanol RP-HPLC step (Table1, third step), chromophore markers corresponding to natriureticallyactive materials could be identified when processing different batchesof urine. By rechromatographing fractions 38-58 and 63-66 using amodified acetic acid-methanol method (Table 1, fourth step) employing ashorter gradient, the two natriuretically active marker chromophores,designated LLU-α and LLU-γ, reproducibly eluted at 27.8 and 35.4minutes, respectively. This fourth purification step allowed consistentidentification of natriuretically active crude isolates.

[0154] The LLU-α natriuretic isolate was subjected individually tonormal phase chromatography on silica gel (Beckman Ultrasphere, 5 μm,10×250 mm) eluting at 2 mL/minute with a hexane (B) isopropanol (A)gradient (6% A:94% B for 25 minutes, a linear gradient to 100% A over 30minutes, 100% A for 20 minutes, a linear gradient to 6% A:94% B over 5minutes and an equilibration period at 6% A:94% B for 35 minutes).Seventy (70) one-minute fractions were collected from this fifthpurification step (Table 1). Fluorescence was monitored as describedabove. The wavelength monitored for each of the isolates was selectedbased upon its absorbance spectrum from the prior chromatogram.Chromatography of the first Isolate (LLU-α) was monitored at 295 nm andthat of the second (LLU-γ) at 267 nm. Fractions exhibiting UV absorbancecharacteristic of LLU-α and LLU-γ were bioassayed (see below).

[0155] 2. Extraction Method

[0156] Freeze-dried material obtained from the gel filtrationchromatography was stirred with 9 volumes of isopropanol for 18 hours.The isopropanol solution was then removed and evaporated to dryness on arotary evaporator under reduced pressure. The resulting thick, darkbrown oil from the isopropanol soluble phase was weighed and thenalternately stirred and sonicated for 6 hours and finally stirred for anadditional 18 hours, with 10 volumes of diethyl ether. The ethersolution was then decanted and 4 volumes of ether were added to theremaining insoluble material. After stirring for 72 hours, the ethersolution was again decanted. Two volumes of deionized distilled waterand 2 volumes of diethyl ether were added to the residue. After stirringfor 2 hours, the ether phase was separated and the aqueous phase waswashed three times with one volume of ether. The combined ether extractswere washed with saturated aqueous NaCl and water, and taken to drynesson a rotary evaporator under reduced pressure. The residue wasredissolved in 95% ethanol and again taken to dryness.

[0157] The ether extraction residue was dissolved in 40% aqueousmethanol and subjected to acetic acid-methanol RP-HPLC (Table 1, thirdstep). The chromatographic region from LLU-α to LLU-γ, as identified bytheir characteristic UV spectra, was pooled, dried, re-suspended andchromatographed on the second modified acetic acid-menthol RP-HPLC(Table 1, fourth step). Only LLU-α and LLU-γ were detected after thischromatography step.

[0158] Isocratic acetic acid-methanol RP-HPLC (Table 1, fifth step) wasthen performed on LLU-α. Employing a Beckman Ultrasphere ODS (C₁₈)column (5 μm; 10×250 mm), LLU-α was eluted at 2 mL/minute with 45% 0.2 Macetic acid and 55% methanol for 35 minutes collecting seventy (70)half-minute fractions. The eluant was monitored for absorbance at 290 nm(diode array) and fluorescence. LLU-α was identified by its UV spectrumand subjected to silica gel HPLC (Table 1, sixth step).

[0159] The fractions containing LLU-α from the silica gel HPLC werepooled and subjected to another C₁₈ RP-HPLC step. In this seventhpurification step (Table 1), a Beckman Ultrasphere ODS column (5 μm;4.6×250 mm) was eluted at 1 mL/minute with a gradient of 50 mM aceticacid (A) and 45 mM acetic acid in acetonitrile (B) (85% A:1 5% B for 3minutes, a linear gradient to 100% B over 42 minutes, 100% B for 5minutes). The column was washed with 1:1 methylene chloride:acetonitrile for 5 minutes followed by re-equilibration at initialconditions for 16 minutes. Chromatography was monitored at 265 and 295nm with the diode array detector. Fifty (50) half-minute fractions werecollected staring at 10 minutes.

[0160] The extraction purification procedure increased the yield ofisolated LLU-α by about 50%. In the chromatographic procedure,encompassing a total of five purification steps, less than 1 mg of LLU-αwas obtained from about 105 g of lyophilized G-25 material (yield lessthan 9×10⁻⁴%). Approximately 1.8 mg of LLU-α resulted from theextraction procedure (seven purification steps) applied to about 155 gof lyophilized G-25 product (yield approximately 1.2×10⁻³%) . The twoadditional RP-HPLC steps of this procedure led to essentially pureLLU-α. Likewise, the yield of LLU-α appeared to increase comparably.

[0161] LLU-α from the modified acetic acid-methanol RP-HPLCchromatography step (Table 1, fourth step) can be further purified usinga method compatible for LC-MS. In this purification step, a BeckmanUltrasphere ODS column (5 μm, 4.6×250 mm) was eluted isocratically at 1mL/minute with 0.1% trifluoroacetic acid, 40% acetonitrile, and 60%water for 30 minutes. LLU-γ from the previous chromatographic stepelutes at 16.5 minutes. Between runs the column is washed with 0.1%trifluoroacetic acid in acetonitrile for 10 minutes, followed byreequilibration at initial conditions for 10 minutes. Chromatography wasmonitored at 265 and 230 nm with a diode array detector. LLU-γ wascollected as a single fraction. TABLE II Chemical characteristics of thenatriuretic LLUs LLU-α LLU-γ Exact Mass 264.1373 ND^(a) EmpiricalC₁₅H₂₀O₄ ND Formula UV λmax 205 nm λmax 220 nm Characteristics λmax 294nm λmax 268 nm Functional carboxyl ND Groups Determined hydroxyl by IRaryl ether Physical Unstable in Unstable when Properties dilute BasePurified Unstable in Very Unstable in CDCl₃ Dilute Base Reaction withHNF-α methyl ester ND CH₂N₂ C₁₄H₁₉O₂CO₂CH₃ MW 278.1515 + Other Products

[0162] Isolated from early fractions of silica gel HPLC of LLU-α was thedrug naproxen, which was being administered to some urine donors. Itsidentity was determined by NMR and verified by comparison with the NMRspectrum of commercial naproxen. Naproxen serves as an additional markerduring the silica gel HPLC.

[0163] 3. Treatment of LLU-α with CH₂N₂

[0164] Diazomethane was generated by treatment of1-methyl-3-nitro-1-nitrosoguanidine (112 mg, 760 μmol) with 400 μL 50%KOH (aq). The diazomethane was distilled into 1 mL diethyl ether at −7°C. This solution was then added to 700 μg (2.6 μmol) LLU-α in 0.5 mLdiethyl ether at 0° C. The reaction mixture was warmed to ambienttemperature, then allowed to stand for 40 minutes. Solvent was removedunder a stream of N₂ and the residue dissolved in 15% 45 mM acetic acidin acetonitrile/85% 50 mM acetic acid and subjected to the aceticacid-acetonitrile RP-HPLC purification step as described above (seventhstep). The approximate yield of the ester was 53%. Methyl esterificationof LLU-α followed by RP-HPLC yielded essentially pure LLU-α methylester. The methyl ester was synthesized to further the characterizationof LLU-α. LLU-α methyl ester eluted as an apparently homogenous singlepeak from acetic acid—acetonitrile RP-HPLC. A total of approximately 0.9mg of LLU-α methyl ester was isolated and subjected to chemicalcharacterization by ultraviolet, infrared, ¹³C- and ¹H-NMR and massspectroscopy. The physical chemical characteristics, molecular weightand inferred molecular formula of both LLU-α and its methyl ester arelisted in Table II.

Bioassays for Biological Activity EXAMPLE 2

[0165] 1. In Vivo Bioassay

[0166] The assay for natriuresis in conscious rats has been describedpreviously (see Benaksas et al., above). The assay is briefly reiteratedhere. Female Sprague-Dawley (Harlan) rats (200-250 g) were cannulated inthe femoral artery and vein for monitoring of mean arterial pressure(MAP) and Infusion of saline and samples, respectively. The bladder wascatheterized for collection of urine in ten-minute periods. Furosemide(0.4 mg/kg bwt; 1 mg/mL in 0.17% saline) was infused as a positivecontrol at the beginning of the sixth ten-minute period. The sample wasinfused at the beginning of the seventeenth ten-minute period. Urine wascollected for another 150 minutes. The volume of the urine wasdetermined gravimetrically and the Na⁺ and K⁺ concentrations determinedwith a Beckman E2A electrolyte analyzer. From these data the sodiumexcretion values (UNaV) were calculated.

[0167] The natriuretic response of a sample was normalized to the doseof furosemide infused. The not sodium excretion for the Infusion offurosemide or sample was calculated as follows. The median sodiumexcretion value (μmoles Na⁺/10 minute period) for the five periodsbefore infusion of furosemide or sample was used to establish a baselinevalue for the calculation of ΔUNaV (=μmoles Na⁺ period−baseline μmolesNa⁺) for administration of either furosemide or sample respectively. Thesum of ΔUNaV for the four periods following infusion of furosemide wasthe net sodium excreted for furosemide, defined as FR. The sum of ΔUNaVfor the fifteen periods following infusion of the sample was the netsodium excreted for the sample defined as SR. The natriuretic ratioR_(n) (or normalized natriuretic response) of a sample was calculated bydividing SR by FR (R_(n)=SR/FR). A sample is considered natriureticallyactive if the R_(n) value for that sample was greater than or equal to0.67 (greater than 99% confidence limits).

[0168] Partially purified LLU-α from silica gel-HPLC (sixth purificationstep) was assayed for natriuretic activity utilizing the in vivobioassay. It was active in the 4-8 μg/kg dose range and showed noactivity at lower or higher doses (Table III). LLU-α is also active at 8μg/kg when evaluated in the in vivo bioassay after being furtherpurified on acetic acid/acetonitrile RP-HPLC (seventh step of extractionmethod). TABLE III Dose response of LLU-α present in fractions from thesilica gel HPLC step of the extraction procedure from uremic urineFraction Dose (μg) Natriuretic Response (R)^(a) 17 0.2 −0.14 1 0.27 21.14 2 0.75 10 0.26 18 56.4 0.23 22.4 0.02 19 0.2 0.24 1 0.93 2 −0.10 20.82 10 0.09 20 2 1.32 2 0.39 21 2 −0.06 2 0.39

[0169] LLU-α and -β when infused into the rat produced sustainednatriuresis with no effect on blood pressure. LLU-γ has not beenpurified sufficiently to obtain a dose-response curve for natriuresis,owing to its instability. LLU-α displays a narrow and biphasicnatriuretic dose-response curve (Table III). There was no detectablekaliuresis when LLu-α as infused. Some kaliuresis occurred after theinfusion of LLU-γ, as not always observed. Neither LLU-α nor -γ caused asignificant arterial pressure.

[0170] 2. Na⁺/K⁺-ATPase Inhibition Assay

[0171] the assay in MDBK cells has been described previously (seeBenaksas at al., above). The assay is described briefly here.Madin-Darby bovine kidney (MDBK) cells (ATCC:CCL22) were maintained inDulbecco's Modified Eagle's Medium (DMEM) with 5% Fetal Bovine Serum and5% Bovine Calf Serum in a 5% CO₂/95% humidified air atmosphere at 37° C.and split (1:2) once per week.

[0172] One day before the assay, cells were plated in a 96-well plate ata density of 5×10⁵ cells/well in DMEM with serum. On the day of theassay the medium was removed and the cells washed with phosphatebuffered saline (PBS) before addition of 100 μL of assay media (122 mMNaCl, 1.8 mM CaCl₂, 0.8 mM MgSO₄, 24 mM NaHCO₃, 1 mM sodium pyruvate, 25mM glucose, 14 mM glycylglycine, 0.2% phenol red, 8 mM Na₂HPO₄ 1.15 mMKH₂PO₄, pH 8.0) and 100 μl of sample. The plate was preincubated for 30minutes at 37° C., then chilled on ice for 10 minutes. To each well wasadded 0.15 μCi ⁸⁶RbCl (Amersham) in 10 μL of assay media. The plate wasthen incubated at 37° C. for 10 minutes. A portion (100 μL) of thesupernatant was counted with 0.5 mL of scintillation cocktail in aliquid scintillation counter. As a control for Na⁺/K⁺-ATPase inhibition,a dose response curve for ouabain in the range of 10⁻⁵-10⁻⁸ M wasobtained. Intra-experiment coefficient of variation for ouabain was3-15%. Inhibition of ⁸⁶Rb⁺ uptake by samples was corrected for thatuptake which was inhibitable by ouabain.

[0173] When LLU-α was assayed in the Na⁺/K⁺-ATPase inhibition assay itexhibited no inhibition in the range of 0.2-200 ng/well. Assay of crudeLLU-γ obtained from the acetic acid-methanol RP-HPLC rechromatographystep in the sodium pump inhibition assay showed no inhibition of thesodium pump.

Analytical Spectoscopy EXAMPLE 3

[0174] In addition, spectroscopy other than UV was performed. ¹³C- and¹H-NMR spectra were recorded at 500.1357 MHz in deutero-Chloroform(99.9%) in a GN-500 spectrometer (General Electric). High resolutionElectron-Impact (ED mass spectra with a resolution of 2000 were recordedat an ionization voltage of 70 eV, source temperature of 220° C. andintroduction of sample by direct probe on a VG7070 EHF high resolutionmass spectrometer. Fourier-transform infrared (FT-IR) spectroscopy wasperformed on a Nicolet 5DX with 4 wavenumber resolution.

[0175] The IR and ¹³C-NMR spectra of LLU-α provided evidence for thepresence of a carboxylic acid group. This explained the tailing of LLU-αobserved upon elution from isopropanol/hexane silica gel HPLC (sixthpurification step). The presence of a carboxyl group was verified whenthe reaction of LLU-α with diazomethane resulted in a product that wasless polar on RP-HPLC and had an exact mass 14 units greater than LLU-αas determined by MS (Table II). This is consistent with the formation ofa methyl ester.

[0176] In the following synthesis examples, Examples 4-10 set forthgeneral methods useful to produce a wide range of compounds within thescope of the invention. Examples 11-21 describe syntheses of specificcompounds.

Synthesis 0f Racemic 6-hydroxychromans EXAMPLE 4

[0177]

[0178] To a solution of hydroquinone 1 (0.01 mol) and a catalyst,preferably boron trifluoride diethyl etherate (0.016 mol) In an organicsolvent, preferably dry dioxane (10 mL), is added vinyl lactone 2 (0.016mol) in an organic solvent, preferably dry dioxane (5.0 mL) over 1-60minutes, preferably 60 minutes, at 0-150° C., preferably 110° C., underan inert gas. The reaction mixture is stirred for 0 to 8 hours,preferably 0 hours, at the selected temperature, cooled to roomtemperature, and diluted with an organic solvent, preferably diethylether (200 mL) The reaction mixture is then washed with water (100 mL,2×50 mL), dried over sodium sulfate (Na₂SO₄), and solvent is removedunder reduced pressure to afford a brown oily residue. The residue isdissolved in alcohol, preferably methanol (30 mL), and the alcohol isthen removed under reduced pressure. The brown oily liquid or semisolidis further purified by chromatography, preferably on silica gel, toafford pure racemic chroman derivative 3.

Synthesis of Racemic Chromans EXAMPLE 5

[0179]

[0180] To a solution of phenol 4 (0.01 mol) and a catalyst, preferablyboron trifluoride diethyl etherate (0.016 mol) in an organic solvent,preferably dry dioxane (10 mL), Is added vinyl lactone 2 (0.016 mol) Inan organic solvent, preferably dry dioxane (5.0 mL) over 1-60 minutes,preferably 60 minutes, at 0-150° C., preferably 110° C., under an inertgas. The reaction mixture is stirred for 0 to 8 hours, preferably 0hours, at the selected temperature, cooled to room temperature, anddiluted with an organic solvent, preferably diethyl ether (200 mL). Thereaction mixture is then washed with water (100 mL, 2×50 mL), dried oversodium sulfate (Na₂SO₄), and solvent is removed under reduced pressureto afford a brown oily residue. The residue is dissolved in alcohol,preferably methanol (30 mL), and the alcohol is then removed underreduced pressure. The brown oily liquid or semisolid is further purifiedby chromatography, preferably on silica gel, to afford pure racemicchroman derivative 5.

Synthesis of Chroman Methyl Esters EXAMPLE 6

[0181]

[0182] Chroman 3 (R₃═OH) or 5 (see Examples 4 and 5 above) (0.01 mol) isdissolved in methanol (30 mL), and a solution of diazomethane in etheris added at 0-5° C. until the yellow color of the diazomethane remains.The reaction mixture is left at room temperature for 2-5 hours, solventis removed, and the desired product 6 is crystallized from a suitableorganic solvent.

Syntheisis of Chroman Esters EXAMPLE 7

[0183]

[0184] Chroman 3 (R₃═OH) or 5 (10 mmol) is dissolved in drytetrahydrofuran (30 mL) with an alcohol R₇—OH (12 mmol),1-hydroxybenzotriazole (10 mmol) and 1,3-dicyclohexylcarbodilmide (11mmol) at 2-5° C. The reaction mixture is stirred at 2-5° C. for one hourand at 23° C. for one to 20 hours. Precipitated dicyclohexyl urea isfiltered, solvent is removed under reduced pressure, and the residue isdiluted with ethyl acetate (150 mL). The organic phase is washed withaqueous KHSO₄ (10%, 40 mL), water (50 mL) and saturated aqueous hydrogencarbonate (50 mL), and then dried over sodium sulfate. The solvent isremoved under reduced pressure, and the residue is purified bychromatography, preferably silica gel, to afford pure racemic ester 7.

Synthesis of Chroman Amides EXAMPLE 8

[0185]

[0186] Chroman 3 (R₃═OH) or 5 (10 mmol) is dissolved in drytetrahydrofuran (40 mL), and neutralized with N-methylmorpholine,isobutyl chlorocarbonate (10 mmol) is added, followed one minute laterby a selected amine (R₇—NH₂ or R₇R₈—NH), or ammonia (11 mmol). Thereaction mixture is allowed to reach room temperature. After stirring atroom temperature for 1 hour, THF is removed under reduced pressure, andthe residue is taken into ethyl acetate (250 mL). The ethyl acetatesolution is successively washed with aqueous KHSO₄ (10%, 40 mL), water(50 mL), and saturated aqueous hydrogen carbonate (50 mL), and thendried over sodium sulfate. The solvent is removed under reducedpressure, and the residue is purified by chromatography, preferablysilica gel, to afford pure racemic amide 8.

Synthesis of R₄ Chroman Esters EXAMPLE 9

[0187]

[0188] Method 1: Chroman 3 (R₃═OH) or 5 (10 mmol) is dissolved inpyridine (20 mL), and acid anhydride (30 mmol) is added at 5° C. Thereaction mixture is left at room temperature for 18 hours, solvent isremoved in vacuum, and the residue is dissolved in ethyl acetate (100mL), washed with citric acid (10%, 30 mL). and water (30 mL), and driedover sodium sulfate. The solvent is removed and the residue iscrystallized from ethyl acetate/hexane to afford ester 9.

[0189] Method 2: Chroman 3 (R₃═OH) or 5 (10 mmol) is dissolved in drypyridine (50 mL) under nitrogen and cooled in an ice-water bath. Acylchloride (10 mmol) is added via syringe over 15 minutes. Stirring iscontinued for 1 hour at room temperature. Pyridine is removed underreduced pressure, and the residue is dissolved in ethyl acetate (100mL). The ethyl acetate phase is washed with water (2×40 mL), aqueoushydrochloric acid (0.05 M, 30 mL) and water (40 mL), and dried oversodium sulfate. The solvent is removed under reduced pressure, and theresidue is purified by chromatography, preferably on silica gel, toafford ester 9.

Synthesis of Oxidized Chroman Derivatives EXAMPLE 10

[0190]

[0191] Chroman 3 or 5 (0.3 mmol) is dissolved in methanol (2.5 mL) in aflask. A ferric chloride solution is prepared by dissolving 1.0 gFeCl₃-6H₂O in water (4.0 mL) and adding methanol (4.0 mL) The ferricchloride solution (2.5 mL) Is added to the flask at room temperaturewith vigorous stirring for 30 minutes in darkness. Methanol 13 removedin vacuum, and the residue Is dissolved In ether (70 mL) The ethersolution is washed with water (3×20 mL) and dried over sodium sulfate,then the solvent is removed. The product is purified on an RP HPLCcolumn (CH₃CH/H₂O gradient) to afford a yellow-to-brown oily product.

Synthesis of Racemic 2,7,8-rimethyl-2-(β-carboxyethyl)-6-hydroxy Chroman(LLU-α) EXAMPLE 11

[0192]

[0193] To a solution of 2,3-dimethyl-1,4-hydroquinone (0.01 mol) andboron trifluoride diethyl etherate (0.016 mol) in dioxane (10 mL, driedon sodium) in a flask was added γ-methyl-γ-vinylbutyrolactone (0.016mol) in dioxane (5.0 mL) over 50 min at 110° C. (oil bath, reflux) undernitrogen. The reaction mixture was cooled to room temperature anddiluted with ether (200 mL), then washed with water (100 mL, 2×50 mL)and dried over sodium sulfate. Ether was then removed under reducedpressure to afford a brown, oily residue. The residue was dissolved inmethanol (30 mL) and solvent was removed under reduced pressure. Theresidue was redissolved in methanol (10 mL) and the flask was purgedwith nitrogen and stored at 5° C. for 20 hours. The resulting suspensionwas centrifuged, and the supernatant was removed. The remaining whitesolid (see Example 21, below) was suspended in aqueous 70% methanol (15mL) and again centrifuged. The supernatant was combined with theprevious supernatant, and methanol was removed in vacuum to afford abrown, oily liquid. The liquid was further purified by flash columnchromatography on silica gel (eluent ethyl acetate/hexane/acetic acid,500:300:1) to afford pure racemic2,7,8-trimethyl-2-(β-carboxyethyl)-6-hydroxy chroman, which wascrystallized from ether-hexane in a yield of 40%. M.P.: 147-148° C.

Synthesis of Racemic 2,5,7,8-tetramethyl-2-(β-carboxyethyl)-6-hydroxyChroman EXAMPLE 12

[0194]

[0195] To a solution of 2,3,5-trimethyl-1,4-hydroquinone (0.01 mol) andboron trifluoride diethyl etherate (0.016 mol) in dioxane (10 mL, driedon sodium) in a flask was added γ-methyl-γ-vinylbutyrolactone (0.016mol) in dioxane (5.0 mL) over 50 min at 110° C. (oil bath, reflux) undernitrogen. The reaction mixture was cooled to room temperature anddiluted with ether (200 mL), then washed with water (100 mL, 2×50 mL)and dried over sodium sulfate. Ether was then removed in vacuum. Theresidue was dissolved in methanol (30 mL), and solvent was removed invacuum. The brown, oily residue was dissolved in methanol (20 mL), andwater was added until the solution became turbid (app. 20 mL), then theflask was purged with nitrogen and stored overnight in a refrigerator.The light yellow solid was filtered on a sinter funnel, washed withaqueous 50% methanol and dried in a dessicator. The product was furtherpurified by flash column chromatography on silica gel (eluent ethylacetate/hexane/acetic acid, 500:300:1) to afford pure racemic2,5,7,8-tetramethyl-2-(β-carboxyethyl)-6-hydroxy chroman, which wascrystallized from ether-hexane in a yield of 50%. M.P.: 173° C.

Synthesis of Racemic 2,5,7,8-tetramethyl-2-(β-carboxyethyl)-ChromanEXAMPLE 13

[0196]

[0197] To a solution of 2,3,5-trimethylphenol (0.01 mol) and borontrifluoride diethyl etherate (0.016 mol) in dioxane (10 mL, dried onsodium) in a flask was added γ-methyl-γ-vinylbutyrolactone (0.016 mol)in dioxane (5.0 mL) via syringe pump over 50 min at 110° C. (oil bath,reflux) under nitrogen. The reaction mixture was cooled to roomtemperature and diluted with ether (200 mL), then washed with water (100mL, 2×50 mL) and dried over sodium sulfate. Ether was then removed invacuum. The residue was dissolved in methanol (30 mL) and solvent wasremoved in vacuum. The reaction mixture was purified by flash columnchromatography on silica gel (eluent ethyl acetate/hexane, 1:1).Fractions containing the desired chroman were pooled, solvent wasremoved, and the compound was crystallized from ethyl acetate/hexane toafford a white crystalline product in a yield of 40%. M.P.: 148-149° C.

Synthesis of Racemic 2,7,8-trimethyl-2-(β-carboxyethyl)-chroman EXAMPLE14

[0198]

[0199] To a solution of 2,3-dimethylphenol (0.01 mol) and borontrifluoride diethyl etherate (0.016 mol) in dioxane (10 mL, dried onsodium) in a flask was added γ-methyl-γ-vinylbutyrolactone (0.016 mol)in dioxane (5.0 mL) via syringe pump over 50 min at 110° C. (oil bath,reflux) under nitrogen. The reaction mixture was cooled to roomtemperature and diluted with ether (200 mL) then washed with water (100mL, 2×50 mL) and dried over sodium sulfate. Ether was then removed invacuum. The residue was dissolved in methanol (30 mL), and solvent wasremoved in vacuum. The reaction mixture was purified by flash columnchromatography on silica gel (eluent ethyl acetate/hexane, 1:1).Fractions containing the desired chroman were pooled, solvent wasremoved, and the compound was crystallized from ethyl acetate/hexane.M.P.: 93-94° C.

Synthesis of Racemic 4-methyl-6-(5,6-dimethylbenzohinoyl)-4-hexanolidEXAMPLE 15

[0200]

[0201] Racemic 2,7,8-trimethyl-2-(β-carboxyethyl)-6-hydroxychroman (100mg) was dissolved in methanol (2.5 mL) in a flask. A solution of ferricchloride was prepared by dissolving 1.0 g FeCl₃-6H₂O in water (4.0 mL)and adding methanol (4.0 mL) The ferric chloride solution (2.5 mL) wasadded to the flask at room temperature with vigorous stirring indarkness for 30 minutes. Methanol was removed in vacuum, and the residuewas dissolved In ether (70 mL). The ether solution was washed with water(3×20 mL) dried over sodium sulfate, and the solvent was removed. Theproduct was purified on an RP HPLC column (CH₃CN/H₂O gradient) to afforda yellow-to-brown oily product in 60% yield.

Synthesis of Racemic 4-methyl-6-(3,5,6-trimethybenzochinoyl)-4-hexanolidEXAMPLE 16

[0202]

[0203] Racemic 2,5,7,8-tetramethyl-2-(β-carboxyethyl)-6-hydroxychroman(100 mg) was dissolved in methanol (2.5 mL) in a flask. The ferricchloride solution of Example 10 (2.5 mL) was added to the flask at roomtemperature with vigorous stirring in darkness for 30 minutes. Methanolwas removed in vacuum, and the residue was dissolved in ether (70 mL)The ether solution was washed with water (3×20 mL) dried over sodiumsulfate, and the solvent was removed. The product was purified on an RPHPLC column (CH₃CN/H₂O gradient) to afford a yellow-to-brown oilyproduct in 60% yield.

Resolution of Racemic 2,7,8-trimethyl-2-(β-caboxyethyl)-6-hydroxyChroman (LLU-α) EXAMPLE 17

[0204] The resolution of (S) and (R)-enantiomers was carried out on an(S,S)-WHELK-O 1 column (Regis Technologies, Inc.) 250×4.6 mm, 1 mL/min,using as eluent isocratic 80% hexane:20% propanol:0.5% acetic acid.Fractions were monitored by UV spectroscopy, collected and dried underan argon stream. The enantiomers elute at 6.8 minutes and 8.7 minutes.Isolated LLU-α, when run on this system, elutes at 8.6 minutes.

Synthesis of (R)-and (S)-4-methyl-6-(5,6-dimethylbenzochiol)-4-hexanolidEXAMPLE 18

[0205]

[0206] (R)-2,7,8-Trimethyl-2-(β-carboxyethyl)-6-hydroxy chroman 0 00 mg)(see Example 17) was dissolved in methanol (2.5 mL) and ferric chloridesolution (2.5 mL) was added at room temperature with vigorous stirringfor 30 minutes in darkness. Methanol was removed under reduced pressure,and the residue was dissolved in ether (70 mL). The ether solution waswashed with water (3×20 mL) dried over sodium sulfate, and the solventwas removed. The product was purified by HPLC, using a Phenomenex column(SPHEREX 10 ODS, 250×21.2 mm) with CH₃CN-H₂O 50:50 for 5 minutes, lineargradient to CH₃CN-H₂O 90:10 in 30 minutes, linear gradient to 100% CH₃CNin 5 minutes, flow rate 6 mL/min. Fractions containing the desiredoxidation product were identified by UV spectroscopy. The fractions werepooled, and solvent was removed under reduced pressure to afford(R)-4-Methyl-6-(5,6-dimethylbenzochinoyl)-4-hexanolid as a yellow tobrown oil.

[0207] The foregoing process was repeated using(S)-2,7,8-Trimethyl-2-(β-carboxyethyl)-6-hydroxy chroman (100 mg) toafford (S)-4-Methyl-6-(5,6-dimethylbenzochinoyl)-4-hexanolid as a yellowto brown oil.

Synthesis of Racemic 2,7,8-trimethyl-2-(β-carboxyethyl)-6-acetyl ChromanEXAMPLE 19

[0208]

[0209] Racemic 2,7,8-Trimethyl-2-(β-carboxyethyl)-6-hydroxy chroman (500mg) (see Example 11) was dissolved in pyridine (20 mL) at roomtemperature, and acetic anhydride (10 mL) was added. The solution wasmaintained at room temperature for 5 hours, solvent was removed undervacuum, methanol (4×10 mL) was added and then removed under reducedpressure. The residual oil was dissolved in ethyl acetate (150 mL) andthe organic phase was washed with water (50 mL) aqueous HCl (1 N, 50 mL)and water (50 mL) then dried over sodium sulfate. Solvent was thenremoved, and the residual oily material was purified on -a silica gelcolumn with hexane/ethyl acetate (1:1). The desired product crystallizedfrom acetone/hexane, m.p. 105-107° C.

Synthesis of Racemic 2,7,8-trimethyl-2-(β-carboxyethyl)-6-acetyl ChromanMethyl Ester EXAMPLE 20

[0210]

[0211] Racemic 2,7,8-Trimethyl-2-(β-carboxyethyl)-6-acetyl chroman (500mg) (see Examples 11 and 19) was dissolved in methanol (10 mL), andetheral diazomethane was added until the yellow color of diazomethaneremained. The solution was maintained at room temperature for 1 hour,solvent was removed, and the residue was purified on a silica gel columnwith hexane/acetone (3:1). The desired product crystallized frommethanol/water, m.p. 87-88° C.

Synthesis of Benzodipyran Methyl Ester EXAMPLE 21

[0212]

[0213] Benzodipyran derivative 10 (m.p. 225-227° C.) was isolated as areaction byproduct from the synthesis of LLU-α (Example 11). Derivative10 exists as a racemic mixture of a meso-(R, S) compound and adiastereomeric pair (R, R) and (S, S). Derivative 10 (1.0 g) wassuspended in methanol (10 mL), and etheral diazomethane was added untilthe yellow color of diazomethane remained. The clear solution wasmaintained at room temperature for 1 hour, solvent was removed, and theresidue was purified on a silica gel column with hexane/acetone (3:1).The desired product crystallized from hexane, m.p. 75-76° C.

Treatment and Prevention of High Blood Pressure EXAMPLE 22

[0214] High blood pressure is a major contributory factor tocardiovascular related illness. The administration of a supplementaccording to the present invention, as detailed in the followingexample, will treat and prevent high blood pressure.

[0215] The blood pressure of a patient suffering from high bloodpressure is determined by conventional methods. The patient is thengiven a daily dose of supplement (200-400 mg) containing a formulationof γ-tocopherol 75% (weight to weight) and LLU-α 25% (weight to weight).The daily course of supplementation is continued for a period of 9-12months after which time the patient's blood pressure is againdetermined. After a period of 12 months, a reduction in blood pressureis observed. As a control, placebos or supplements containing equivalentamounts of α-tocopherol are provided to patients suffering from highblood pressure. The results of this study will demonstrate thatsupplementation with a formulation of γ-tocopherol and LLU-α will treatand prevent, high blood pressure in a patient suffering from thisdisease to a greater extent than supplementation with a placebo or anequivalent amount of α-tocopherol.

Treatment and Prevention of Thromboembolic Disease EXAMPLE 23

[0216] Thromboembolic disease is a considerable problem forinsulin-dependant diabetics, the elderly, and people suffering fromcardiovascular disease. The administration of a supplement of thepresent invention, according to the example below, will treat andprevent thromboembolic disease.

[0217] Blood from a patient suffering from thromboembolic disease isdrawn and a platelet aggregation assay, as known by one of skill in theart, is performed on the sample. (See Richardson and Steiner, Adhesionof Human Platelets Inhibited by Vitamin E, Chapter 24, Vitamin E inHealth and Disease, Packer and Fuchs editors, Marcel Dekker Inc.Publishers 1993 pp. 297-311). The patient is then given a daily dose ofsupplement 200-400 mg containing a formulation of γ-tocopherol 75%(weight to weight) and LLU-α 25% (weight to weight). The daily course ofsupplementation is continued for a period of 2-4 weeks after which timethe patient's blood is again drawn and platelet aggregation isdetermined. After a period of 4 weeks, a reduction in plateletaggregation will be observed. As a control, placebos or supplementscontaining equivalent amounts of α-tocopherol are provided to patientssuffering from thromboembolic disease. The results of this study willdemonstrate that supplementation with a formulation of γ-tocopherol andLLU-α will reduce platelet aggregation and thereby treat and preventthromboembolic disease in a patient suffering from this malady betterthan supplementation with a placebo or an equivalent amount ofα-tocopherol.

Reduction of Platelet Binding to Adhesive Proteins EXAMPLE 24

[0218] Platelet aggregation and thromboembolic disease are related tothe aberrant binding of platelets to adhesive proteins. By following theexample disclosed below, platelet binding to adhesive proteins can beinhibited by supplementation of γtocopherol and LLU-α.

[0219] Blood from a patient suffering from thromboembolic disease isdrawn and a platelet adhesion assay, as known by one of skill in theart, is performed on the sample. (See Richardson and Steiner, Adhesionof Human Platelets Inhibited by Vitamin E, Chapter 24 Vitamin E inHealth and Disease, Packer and Fuchs editors, Marcel Dekker Inc.Publishers 1993 pp. 297-311). The patient is then given a daily dose ofsupplement 100-200 mg containing a formulation of γ-tocopherol 75%(weight to weight) and LLU-α 25% (weight to weight). The daily course ofsupplementation is continued for a period of 2-4 weeks after which timethe patient's blood is again drawn and platelet adhesion is determined.After a period of 4 weeks, a reduction in platelet adhesion will beobserved. As a control, placebos and supplements containing equivalentamounts of α-tocopherol can be provided to patients suffering fromthroboembolic disease. The results of this study will demonstrate thatsupplementation with a formulation of γ-tocopherol and LLU-α will reduceplatelet binding to adhesive protein better than supplementation with aplacebo or an equivalent amount of α-tocopherol.

Treatment and Prevention of Atherosclerosis (Arteriosclerosis) EXAMPLE25

[0220] Oxidized LDL is chemoattractant to circulating monocytes andinhibits macrophage mobility in the intima. Indiscriminate uptake ofoxidatively modified LDL by scavenger receptors of macrophages resultsin cholesterol-laden foam cells and fatty-streak formation. Theseevents, and the potential cytotoxicity of oxidized LDL, further promotethe evolution of fatty streaks to a more advanced lesion andcardiovascular disease. In vitro indices of LDL oxidation are known inthe prior art and can be adapted to determine the ability of aformulations of γ-tocopherol and LLU-α to prevent atheroscelerosis andcardiovascular disease. The following example provides one approach bywhich to treat and prevent atheroscelerosis cardiovascular disease.

[0221] Blood from a patient suffering from atheroscelerosis is drawn andthe amount of oxidized LDL present in the sample is determined. (SeeFrei and Ames, Relative Importance of Vitamin E in AntiperoxidativeDefenses in Human Blood Plasma and Low-density Lipoprotein (LDL),Chapter 10 Vitamin E in Health and Disease, Packer and Fuchs editors,Marcel Dekker Inc. Publishers 1993 pp. 131-139). The patient is thengiven a daily dose of supplement 400-800 mg containing a formulation ofγ-tocopherol 75% (weight to weight) and LLU-α 25% (weight to weight).The daily course of supplementation is continued for a period of 2-4weeks after which time the patient's blood is again drawn and the amountof oxidized LDL present in the sample is determined. As a control,placebos or supplements containing equivalent amounts of α-tocopherolare provided to patients suffering from atheroscelerosis. The results ofthis study will demonstrate that supplementation with a formulation ofγ-tocopherol and LLU-α will reduce the level of oxidized LDL in apatient and thereby treat and prevent. atherosclerosis(arteriosclerosis) and cardiovascular disease better thansupplementation with a placebo or an equivalent amount of α-tocopherol.

Treatment and Prevention of Cancer EXAMPLE 26

[0222] The antioxidant and nitrogen scavenger properties of γ-tocopheroland LLU-α can be used to treat and prevent cancer, as described below.The following example is based on an experimental methodology acceptedby those of skill in the art to reflect anti-tumor effects in the humanbody. (See Elson, Impact of Palm Oil on Experimental Carcinogenesis,Chapter 39 Vitamin E in Health and Disease, Packer and Fuchs editors,Marcel Dekker Inc. Publishers 1993 pp. 533-545). Four groups of mice areused in the study: (1) control mice in which tumor formation is notinduced but treatment with a formulation of γ-tocopherol 75% (weight toweight) and LLU-α 25% (weight to weight) is rendered; (2) control micein which tumor formation is induced and treatment is not rendered; (3)experimental mice in which tumor formation is induced and treatment withγ-tocopherol 75% (weight to weight) is rendered; and (4) experimentalmice in which tumor formation is induced and treatment with aformulation of γ-tocopherol 75% (weight to weight) and LLU-α 25% (weightto weight) is rendered. As a further control, mice in which tumorformation is induced are treated with varying concentrations ofα-tocopherol so as to evaluate the relative effectivity of γ-tocopheroland the formulation of γ-tocopherol and LLU-α, as compared toα-tocopherol.

[0223] Mice which receive treatment with γ-tocopherol or a formulationof γ-tocopherol and LLU-α, as described above, are given 20 mg/kg ofsupplement for a period of 2-4 weeks. Tumor cells derived from aspontaneously arising mammary tumor are then injected into the thigharea of the experimental mice to induce tumor formation. Treatment withγ-tocopherol and the formulation of γ-tocopherol and LLU-α is continuedaccording to the protocol above. After 21 days, the mean volume oftumors in the mice is determined and compared. The results of this studywill demonstrate that the mean volume of tumors in the mice treated withγ-tocopherol and the formulation of γ-tocopherol and LLU-α, is less thanthe mean volume of tumors in the control mice in which tumor formationwas induced but γ-tocopherol or the formulation of γ-tocopherol andLLU-α is not administered.

Reduction in the Formation of Free Radicals EXAMPLE 27

[0224] A reduction in the formation of free radicals is thought to beessential to prevent cancer and cardiovascular disease. The followingexample provides an approach to evaluate the efficacy of supplementationwith a formulation of γ-tocopherol and LLU-α for reducing the formationof free radicals.

[0225] Human excretion of breath pentane has been used in a number ofhuman studies as a measure of free-radical reactions. (See Packer etal., Significance of Vitamin E for the Athlete, Chapter 34, Vitamin E inHealth and Disease, Packer and Fuchs editors, Marcel Dekker Inc.Publishers 1993 pp. 465-471). The breath pentane is measured from twogroups of human volunteers. The first group serves as the control forthe study and is not supplemented with a formulation of γ-tocopherol andLLU-α. The second group is supplemented for 2-4 weeks (200-400 mg/day)with a formulation of γ-tocopherol 75% (weight to weight) and 25%(weight to weight) LLU-α. As another control, human volunteerssupplemented with an equivalent amount α-tocopherol can be used. Boththe control and experimental groups are subjected to exhaustive exerciseand a measurement of breath pentane is taken shortly thereafter. Theresults will show that breath pentane, a measure of free-radicalformation in the body, is reduced in humans who received supplementationwith a formulation of γ-tocopherol and LLU-α as compared to a controlgroup which received either no supplementation or supplementation withα-tocopherol.

Treatment and Prevention of Neuropathological Lesions EXAMPLE 28

[0226] Vitamin E deficiency in animals is associated with axonaldystrophy that involves degeneration in the posterior cord and in thegracile and cuneate nuclei. Humans who suffer from malabsorptionsyndromes that are associated with decreased absorption or transport ofvitamin E develop similar neurological symptoms including byporflexia,gait disturbances, decreased sensitivity to vibration and proprioceptionand opthalmoplegia. Neuropathological lesions, including axonaldegeneration of the posterior cord and the gracilis nucleus in humansare comparable with those found in animals deficient in vitamin E. Ratssuffering from vitamin E deficiency can be used to determine thetherapeutic benefits of supplemention with a formulation of γ-tocopheroland LLU-α, according to the following example, for the treatment andprevention of neurological conditions.

[0227] Rats are maintained on a vitamin E depleted diet for a period of8 weeks so that neuropathological lesions are allowed to develop. Onegroup of vitamin E deficient rats is continued on the vitamin E depleteddiet without vitamin E supplementation during the course of the studyand serves as a control. A second control consists of vitamin Edeficient rats maintained on a vitamin E depleted diet but supplementedwith 20 mg/kg of α-tocopherol. The experimental group of vitamin Edeficient rats is treated with either 20 mg/kg of γ-tocopherol or aformulation 75% (weight to weight) of γ-tocopherol and 25% (weight toweight) LLU-α for a period of 2-4 weeks. The rats are then sacrificedand the presence of neuropathological lesions is determined by methodsknown in the art. The results of this study will demonstrate thatsupplementation with a formulation of γ-tocopherol and LLU-α will treatand prevent the formation of neuropathological lesions associated withvitamin E deficiency better than supplementation with α-tocopherol or nosupplementation at all.

Modulation of Immune System Response EXAMPLE 29

[0228] The main role of vitamin E in enhancing immune response isbelieved to involve the prevention of lipid perioxidation of cellmembranes. The rapidly proliferating cells of the stimulated immune andphagocytic systems are particularly prone to perioxidative damage byfree radicals, peroxides, and superoxides. Vitamin E supplementation hasbeen shown to modulate the immune response of mammals as evidenced by areduction in PGE₂ production, an increase in mitogenic response, anincrease in IL-2 production, and the induction of delayed-typehypersensitivity (DTH). (See Meydani and Tengerdy, Vitamin E and ImmuneResponse, Chapter 40, Vitamin E in Health and Disease, Packer and Fuchseditors, Marcel Dekker Inc. Publishers 1993 pp. 549-561). An improvementin immune response after supplementation with a formulation ofγ-tocopherol and LLU-α are determined by measuring the reduction of PGE₂and the increase in IL-2 production in mice, according to the followingexample.

[0229] A first group of mice, the control group, does not receivetreatment with γ-tocopherol or a formulation of γ-tocopherol and LLU-α.To compare the therapeutic benefits of a formulation of γ-tocopherol andLLU-α with α-tocopherol, a control group which receives treatment withα-tocopherol is used. A second group of mice, the experimental group,receives treatment with a formulation of γ-tocopherol and LLU-α.Treatment consists of 40 mg/kg of a formulation of γ-tocopherol 75%(weight to weight) and LLU-α 25% (weight to weight) for a period of 8weeks. Shortly after the treatment phase, the control and experimentalgroups are administered an antigen which illicits an immune response.Next, the PGE₂ production and IL-2 production is determined byconventional methods. The results of this study will demonstrate thatmice which received treatment with a formulation of γ-tocopherol andLLU-α exhibit a lower level of PGE₂ and an increase in IL-2 productionas compared to control mice which received either α-tocopherolsupplementation or no supplementation at all.

[0230] Although the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims. All references cited herein are hereby expressly incorporated byreference.

What is claimed is:
 1. A method of preventing or treating a diseasecaused by oxidation in vivo by administering a pharmacologicallyeffective amount of at least one compound selected from the groupconsisting of: (1)2,5,7.8-tetramethyl-2-(β-carboxyethyl)-6-hydroxychromane, apharmacologically acceptable salt thereof or a pharmacologicallyacceptable hydrate thereof and (2)2,7.8-trimethyl-2-(β-carboxyethyl)-6-hydroxychromane, apharmacologically acceptable salt thereof or a pharmacologicallyacceptable hydrate thereof.
 2. The method of claim 1, wherein thedisease is caused by oxidated low density lipoprotein (LDL).
 3. Themethod of claim 1, wherein the disease is arteriosclerosis.